International Journal of Neuropsychopharmacology, 2015, 1–11 doi:10.1093/ijnp/pyu071 Research Article
research article
Learning Induces Sonic Hedgehog Signaling in the Amygdala which Promotes Neurogenesis and Long-Term Memory Formation Hui-Chi Hung, MS; Ya-Hsin Hsiao, MS; Po-Wu Gean, PhD Institute of Basic Medical Sciences and Department of Pharmacology, College of Medicine, National ChengKung University, Tainan, Taiwan (Drs Hung, Hsaio, and Gean). Correspondence: Po-Wu Gean, PhD, Department of Pharmacology, College of Medicine, National Cheng-Kung University, Tainan, Taiwan 701 (powu@mail. ncku.edu.tw).
Abstract Background: It is known that neurogenesis occurs throughout the life mostly in the subgranular zone of the hippocampus and the subventricular zone of the lateral ventricle. We investigated whether neurogenesis occurred in the amygdala and its function in fear memory formation. Methods: For detection of newborn neurons, mice were injected intraperitoneally with 5-bromo-2’-deoxyuridine (BrdU) 2 h before receiving 15 tone–footshock pairings, and newborn neurons were analyzed 14 and 42 days after training. To determine the relationship between neurogenesis and memory formation, mice were given a proliferation inhibitor methylazoxymethanol (MAM) or a DNA synthesis inhibitor cytosine arabinoside (Ara-C). To test whether sonic hedgehog (Shh) signaling was required for neurogenesis, Shh-small hairpin–interfering RNA (shRNA) was inserted into a retroviral vector (Retro-Shh-shRNA). Results: The number of BrdU+/Neuronal nuclei (NeuN)+ cells was significantly higher in the conditioned mice, suggesting that association of tone with footshock induced neurogenesis. MAM and Ara-C markedly reduced neurogenesis and impaired fear memory formation. Shh, its receptor patched 1 (Ptc1), and transcription factor Gli1 protein levels increased at 1 day and returned to baseline at 7 days after fear conditioning. Retro-Shh-shRNA, which knocked down Shh specifically in the mitotic neurons, reduced the number of BrdU+/NeuN+ cells and decreased freezing responses. Conclusions: These results suggest that fear learning induces Shh signaling activation in the amygdala, which promotes neurogenesis and fear memory formation. Keywords: amygdala, fear memory, neurogenesis, sonic hedgehog
Introduction Conventionally, memory is classified into short-term memory, lasting minutes to hours, and long-term memory, lasting several hours to days; the latter requires new protein synthesis (McGaugh, 2000; Abraham and Williams, 2008; Alberini, 2008; Gold, 2008; Rudy, 2008). In addition, 10–12 h after a one-trial inhibitory avoidance task and light-footshock association, there is a novel protein synthesis and brain-derived neurotrophic
factor (BDNF)-dependent phase of memory persistence lasting more than 7 days (Bekinschtein et al., 2007 2008; Ou et al., 2010). It is thought persistence of memory requires recurrent activation of neuronal activity (Wittenberg et al., 2002), which results in molecular changes that can reset a population of neurons to participate in future episodes of synaptic plasticity by reinforcing the efficacy of synaptic transmission and growth of dendrites
Received: May 30, 2014; Revised: July 11, 2014; Accepted: July 24, 2014 © The Author 2015. Published by Oxford University Press on behalf of CINP. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected]
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2 | International Journal of Neuropsychopharmacology, 2015
and axons (Tolwani et al., 2002; Lamprecht and LeDoux, 2004). However, a single molecular cascade consisting of receptor activation, changes in protein phosphorylation, and synthesis of new proteins may not be sufficient for the persistence of longterm memory. Particularly, the basolateral amygdala (BLA) is necessary for the establishment and/or maintenance of remote fear memory (Poulos et al., 2009). We tested the hypothesis that neurogenesis may be a candidate mechanism contributing to long-term memory formation in the amygdala. Neurogenesis occurs throughout life predominantly in the subgranular zone of the hippocampus and the subventricular zone of the lateral ventricle (Ming and Song, 2005; Balu and Lucki, 2009). Newly-generated neurons in the subventricular zone migrate to the olfactory bulb and play key roles in olfactory memory (Lledo et al., 2006). In the subgranular zone, new cells are differentiated into both neurons and glial cells, and the newborn neurons incorporate to the granule cell layer of the dentate gyrus (Lledo et al., 2006). A growing body of evidence supports the contribution of hippocampal newborn neurons to hippocampus-dependent memories (Shors et al., 2001; Saxe et al., 2006; Imayoshi et al., 2008). It was reported that neurogenesis in the adult hippocampus is required for trace eye blink conditioning (Shors et al., 2001). However, conflicting results also existed, as ablation of hippocampal neurogenesis had no effect on learning, even in hippocampus-dependent memory tasks such as the contextual fear memory task and the Morris water maze task (Shors et al., 2002; Snyder et al., 2005; Zhang et al., 2008). Recently, a unique functional role for newly-born neurons in exerting an antidepressant-like effect has been demonstrated (Snyder et al., 2011). Sonic hedgehog (Shh) is a morphogen which plays a key role in regulating vertebrate organogenesis and numerous other processes in the developing nervous system, including midbrain and ventral forebrain neuronal differentiation and neuronal precursor proliferation (Ericson et al., 1995; Hynes et al., 1995; Chiang et al., 1996; Wechsler-Reya and Scott, 1999; Britto et al., 2002; Ruiz i Altaba et al., 2002). In the present study, we investigated the effect of fear conditioning on the neurogenesis in the amygdala. We found that activation of Shh signaling contributed to amygdala neurogenesis and the formation of fear memory.
any movement except for respiration, which was recorded by camera and analyzed by Freeze Scan software (Med Associates).
Open Field Test The mice were placed in the open field apparatus, made of a Plexiglas box (40 × 40 × 30 cm), and allowed to explore freely for 15 min. The total distance, velocity, and percentage of duration and distance in the central zone were measured by video tracking software (Ethovision).
Ara-C Infusion Mice were anesthetized with chloral hydrate (400 mg/kg, i.p.) and subsequently were mounted on a stereotaxic apparatus. The 26-gauge stainless steel tubing cannulae were implanted bilaterally into the amygdala (anteroposterior, -1.6 mm; mediolateral, ±3.5 mm; dorsoventral, -4.7 mm). After the mice were allowed 7 days to recover, cytosine arabinoside (Ara-C, 1 mM/1μl/ per side; Colon-Cesario et al., 2006) dissolved in saline was infused bilaterally into the amygdala.
MAM Injection MAM (7 mg/kg; Dupret et al., 2005) was injected intraperitoneally once per day for 7 consecutive days, and then fear conditioning was conducted.
BrdU Labeling BrdU (300 mg/kg; Sigma Aldrich) was injected intraperitoneally into the mice 2 h before fear conditioning (Cameron and McKay, 2001; Taupin, 2007; Magavi and Macklis, 2008). Mice were sacrificed at 14 or 42 days after fear conditioning to measure the newborn neurons (Bischofberger, 2007).
Immunofluorecent Staining
All procedures were approved by the Institutional Animal Care and Use Committee of the College of Medicine, National ChengKung University. Four to five mice were housed in each cage, in a temperature-controlled (22 °C) animal colony, and food and water was available ad libitum. They were maintained on a 12 hour light-dark cycle with lights turned on at 07: 00 hours.
Brains were removed after perfusion with saline and 4% paraformaldehyde in phosphate buffered saline (PBS). Sections were pretreated with a saline-sodium citrate buffer at 85°C for 15 min, incubated in 2 N HCl at 37°C for 30 min, and then rinsed in 0.1 M boric acid (pH 8.5) at room temperature for 10 min. They were incubated in a blocking solution (3% bovine serum in PBS) at room temperature for at least 1 h. The sections were incubated with primary antibodies in PBST plus 3% bovine serum at 4°C overnight. After being washed in PBS and Tween 20 (PBST) three times, the mixture of the secondary antibodies was applied to the sections. The sections were placed at room temperature for 2 h, followed by washing three additional times with PBST. Fluorescence signals were detected with a Leica DM 2500 microscope.
Pavlovian Fear Conditioning
Quantitation
Male C57BL/6J mice at 2 months of age were used in this study. The procedure of fear conditioning was described previously (Hsiao et al., 2011). After a 120 s acclimation period, mice were presented with a tone (20 s, 80 dB, 5 kHz) that co-terminated with a foot shock (1 s, 0.7 mA). This tone–foot shock pairing was repeated 15 times with an inter-trial interval of 2 min. Between training sessions, 75% ethanol was used for cleaning. Cuedependent fear memory was assessed at 1, 14, and 42 days after training. Between each test session, 1% acetic acid was used for cleaning. The freezing behavior was defined as the absence of
The total numbers of BrdU-positive cells per section were counted in the amygdala, central amygdala nuclei, and medial amygdala nuclei. BrdU labeling was performed on every second section of 20 μm (microtome setting) in thickness, at least 560 µμm apart, from each mouse. Cells numbers were done by a MetaMorph image analysis system. Quantitative analysis of BrdU-immune fluoresccent-stained sections was done on a Leica DM 2500 microscope equipped with a video camera. One-way analysis of variance (ANOVA) and Newman–Keuls post hoc tests were performed to analyze the multiple comparisons. For double
Materials and Methods Animal Care
Hung et al. | 3
labeling, the colocalization of both BrdU and the markers NeuN, doublecortin (DCX), or Glial fibrillary acidic protein (GFAP) were confirmed on the slices using confocal laser scanning microscopy (Olmypus IX81, FV10-ASW). Triple labeling was performed for further verification of the BrdU positive cell phenotype. 4ʹ,6-diamidino-2-phenylindole (DAPI) nuclear labeling was performed on every second section, and the total number of Shhpositive cells was counted in the amygdala using a Leica DM 2500 microscope. To determine whether fear memory was specific to pairing groups, coronal sections of the amygdala from naive and unpaired groups were processed for Shh with DAPI labeling.
Antibodies for Staining The following primary antibodies were used: mouse monoclonal antibodies for BrdU (1:200, Millipore), NeuN (1:1 000, Millipore), and GFAP (1:1 000, eBioscience); rat monoclonal antibody for BrdU (1:100, Abcam) and Shh (1:100, R&D systems); rabbit polyclonal antibody for Shh (1:500, Millipore) and enhanced GFP (1:1000, Thermo Fisher Scientific); and rabbit monoclonal antibody for doublecortin (1:200, Cell Signaling Technology). The following secondary antibodies were used: Alexa Fluor®594-conjugated Goat-anti-rabbit IgG, Alexa Fluor®594-conjugated Donkey-anti-rat IgG, Alexa Fluor®488conjugated Sheep-anti-mouse IgG, Alexa Fluor®488-conjugated Goat-anti-rabbit IgG, Alexa Fluor®488-conjugated Donkey-antirat IgG, DyLightTM 405-conjugated Sheep-anti-mouse IgG, and DyLightTM 405-conjugated Donkey-anti-rat IgG (all 1:200, Jackson ImmunoResearch). DAPI (4’,6-diamidino-2-phenylindole; 1:1 000, Sigma-Aldrich) dye was used for staining of nuclei.
Western Blot Analysis Basolateral amygdala tissues were lysed by homogenization buffer (50 mM Tris-HCl, pH 7.5, 0.3 M sucrose, 5 mM EDTA), with protease/phosphatase inhibitor cocktail (Roche). Lysates were centrifuged at 12 000 rpm for 30 min. Supernatants were collected, and then protein concentration was measured by a Bradford assay. Proteins (150 μg) were taken and denatured at 95°C for 5 minutes. After electrophoresis via SDSpolyacrylamide gels (6.5–12.5%), proteins were transferred to nitrocellulose membranes. The membranes were blocked with 5% nonfat dry milk for 1 h at room temperature. Western blotting analysis used rabbit anti-Shh (1:3 000, Millipore), rabbit antiPtc1 (1:500, Proteintech Group), and rat anti-Gli1 (1:15 000, R&D systems) antibodies overnight at 4℃, and then incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. Signals were detected using Hyperfilm-ECL (Amersham Biosciences). The intensity was measured using Image-J.
Shh-shRNA Retrovirus Production To specifically knock down Shh in newborn cells, Shh-shRNA or scrambled shRNA was inserted into a pSIREN-RetroQ-ZsGreen1 retroviral shRNA expression vector (Clontech Laboratories, Inc.). The fluorescent ZsGreen1 protein was used as a tag to indicate transfection efficiency. Retroviruses were produced by the GP2-293 packaging cell line that cotransfected with the pSIREN-RetroQZsGreen1 retroviral shRNA expression vector and pVSV-G using Lipofectamine® LTX Reagent (Life Technologies). Retroviruses were harvested at 48 h after transfection, concentrated by Retro-X™ Concentrator (Clontech Laboratories), and purified to yield 1 × 106 transducing units/ml, which were stored at -80 ℃ until use. Viruses were stereotaxically injected into the amygdalas of the mice at the age of 2 months (anteroposterior, -1.6 mm; mediolateral, ±3.5 mm;
dorsoventral, -4.7 mm). The target sequence of Shh-shRNA and scrambled shRNA were described as follows: Shh-shRNA, 5×-CCCGAC ATCA TATTTAAGGAT-3×; and scrambled shRNA, 5×-GCTGTTGGACAG CGAGACCAT-3×.
Statistical Analysis All values in the text were mean ± standard error of the mean. Two-way ANOVA was used to analyze the differences in freezing responses tested at 1, 14, and 42 days after post-training between MAM- and Vehicle-, Ara-C- and Vehicle-, and retroviral vector (Retro-Shh-shRNA)- and Retro-scramble-shRNA-treated mice. Oneway ANOVA and Newman–Keuls post hoc comparisons were used to analyze the differences in BrdU+/NeuN+ cells and protein levels among naïve, unpaired, and paired mice. Unpaired t-tests were used to analyze differences of BrdU+/NeuN+ cells between Shh-shRNAtreated and scrambled control mice, and between MAM-treated and vehicle control mice. The level of significance was p
research article
Learning Induces Sonic Hedgehog Signaling in the Amygdala which Promotes Neurogenesis and Long-Term Memory Formation Hui-Chi Hung, MS; Ya-Hsin Hsiao, MS; Po-Wu Gean, PhD Institute of Basic Medical Sciences and Department of Pharmacology, College of Medicine, National ChengKung University, Tainan, Taiwan (Drs Hung, Hsaio, and Gean). Correspondence: Po-Wu Gean, PhD, Department of Pharmacology, College of Medicine, National Cheng-Kung University, Tainan, Taiwan 701 (powu@mail. ncku.edu.tw).
Abstract Background: It is known that neurogenesis occurs throughout the life mostly in the subgranular zone of the hippocampus and the subventricular zone of the lateral ventricle. We investigated whether neurogenesis occurred in the amygdala and its function in fear memory formation. Methods: For detection of newborn neurons, mice were injected intraperitoneally with 5-bromo-2’-deoxyuridine (BrdU) 2 h before receiving 15 tone–footshock pairings, and newborn neurons were analyzed 14 and 42 days after training. To determine the relationship between neurogenesis and memory formation, mice were given a proliferation inhibitor methylazoxymethanol (MAM) or a DNA synthesis inhibitor cytosine arabinoside (Ara-C). To test whether sonic hedgehog (Shh) signaling was required for neurogenesis, Shh-small hairpin–interfering RNA (shRNA) was inserted into a retroviral vector (Retro-Shh-shRNA). Results: The number of BrdU+/Neuronal nuclei (NeuN)+ cells was significantly higher in the conditioned mice, suggesting that association of tone with footshock induced neurogenesis. MAM and Ara-C markedly reduced neurogenesis and impaired fear memory formation. Shh, its receptor patched 1 (Ptc1), and transcription factor Gli1 protein levels increased at 1 day and returned to baseline at 7 days after fear conditioning. Retro-Shh-shRNA, which knocked down Shh specifically in the mitotic neurons, reduced the number of BrdU+/NeuN+ cells and decreased freezing responses. Conclusions: These results suggest that fear learning induces Shh signaling activation in the amygdala, which promotes neurogenesis and fear memory formation. Keywords: amygdala, fear memory, neurogenesis, sonic hedgehog
Introduction Conventionally, memory is classified into short-term memory, lasting minutes to hours, and long-term memory, lasting several hours to days; the latter requires new protein synthesis (McGaugh, 2000; Abraham and Williams, 2008; Alberini, 2008; Gold, 2008; Rudy, 2008). In addition, 10–12 h after a one-trial inhibitory avoidance task and light-footshock association, there is a novel protein synthesis and brain-derived neurotrophic
factor (BDNF)-dependent phase of memory persistence lasting more than 7 days (Bekinschtein et al., 2007 2008; Ou et al., 2010). It is thought persistence of memory requires recurrent activation of neuronal activity (Wittenberg et al., 2002), which results in molecular changes that can reset a population of neurons to participate in future episodes of synaptic plasticity by reinforcing the efficacy of synaptic transmission and growth of dendrites
Received: May 30, 2014; Revised: July 11, 2014; Accepted: July 24, 2014 © The Author 2015. Published by Oxford University Press on behalf of CINP. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected]
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2 | International Journal of Neuropsychopharmacology, 2015
and axons (Tolwani et al., 2002; Lamprecht and LeDoux, 2004). However, a single molecular cascade consisting of receptor activation, changes in protein phosphorylation, and synthesis of new proteins may not be sufficient for the persistence of longterm memory. Particularly, the basolateral amygdala (BLA) is necessary for the establishment and/or maintenance of remote fear memory (Poulos et al., 2009). We tested the hypothesis that neurogenesis may be a candidate mechanism contributing to long-term memory formation in the amygdala. Neurogenesis occurs throughout life predominantly in the subgranular zone of the hippocampus and the subventricular zone of the lateral ventricle (Ming and Song, 2005; Balu and Lucki, 2009). Newly-generated neurons in the subventricular zone migrate to the olfactory bulb and play key roles in olfactory memory (Lledo et al., 2006). In the subgranular zone, new cells are differentiated into both neurons and glial cells, and the newborn neurons incorporate to the granule cell layer of the dentate gyrus (Lledo et al., 2006). A growing body of evidence supports the contribution of hippocampal newborn neurons to hippocampus-dependent memories (Shors et al., 2001; Saxe et al., 2006; Imayoshi et al., 2008). It was reported that neurogenesis in the adult hippocampus is required for trace eye blink conditioning (Shors et al., 2001). However, conflicting results also existed, as ablation of hippocampal neurogenesis had no effect on learning, even in hippocampus-dependent memory tasks such as the contextual fear memory task and the Morris water maze task (Shors et al., 2002; Snyder et al., 2005; Zhang et al., 2008). Recently, a unique functional role for newly-born neurons in exerting an antidepressant-like effect has been demonstrated (Snyder et al., 2011). Sonic hedgehog (Shh) is a morphogen which plays a key role in regulating vertebrate organogenesis and numerous other processes in the developing nervous system, including midbrain and ventral forebrain neuronal differentiation and neuronal precursor proliferation (Ericson et al., 1995; Hynes et al., 1995; Chiang et al., 1996; Wechsler-Reya and Scott, 1999; Britto et al., 2002; Ruiz i Altaba et al., 2002). In the present study, we investigated the effect of fear conditioning on the neurogenesis in the amygdala. We found that activation of Shh signaling contributed to amygdala neurogenesis and the formation of fear memory.
any movement except for respiration, which was recorded by camera and analyzed by Freeze Scan software (Med Associates).
Open Field Test The mice were placed in the open field apparatus, made of a Plexiglas box (40 × 40 × 30 cm), and allowed to explore freely for 15 min. The total distance, velocity, and percentage of duration and distance in the central zone were measured by video tracking software (Ethovision).
Ara-C Infusion Mice were anesthetized with chloral hydrate (400 mg/kg, i.p.) and subsequently were mounted on a stereotaxic apparatus. The 26-gauge stainless steel tubing cannulae were implanted bilaterally into the amygdala (anteroposterior, -1.6 mm; mediolateral, ±3.5 mm; dorsoventral, -4.7 mm). After the mice were allowed 7 days to recover, cytosine arabinoside (Ara-C, 1 mM/1μl/ per side; Colon-Cesario et al., 2006) dissolved in saline was infused bilaterally into the amygdala.
MAM Injection MAM (7 mg/kg; Dupret et al., 2005) was injected intraperitoneally once per day for 7 consecutive days, and then fear conditioning was conducted.
BrdU Labeling BrdU (300 mg/kg; Sigma Aldrich) was injected intraperitoneally into the mice 2 h before fear conditioning (Cameron and McKay, 2001; Taupin, 2007; Magavi and Macklis, 2008). Mice were sacrificed at 14 or 42 days after fear conditioning to measure the newborn neurons (Bischofberger, 2007).
Immunofluorecent Staining
All procedures were approved by the Institutional Animal Care and Use Committee of the College of Medicine, National ChengKung University. Four to five mice were housed in each cage, in a temperature-controlled (22 °C) animal colony, and food and water was available ad libitum. They were maintained on a 12 hour light-dark cycle with lights turned on at 07: 00 hours.
Brains were removed after perfusion with saline and 4% paraformaldehyde in phosphate buffered saline (PBS). Sections were pretreated with a saline-sodium citrate buffer at 85°C for 15 min, incubated in 2 N HCl at 37°C for 30 min, and then rinsed in 0.1 M boric acid (pH 8.5) at room temperature for 10 min. They were incubated in a blocking solution (3% bovine serum in PBS) at room temperature for at least 1 h. The sections were incubated with primary antibodies in PBST plus 3% bovine serum at 4°C overnight. After being washed in PBS and Tween 20 (PBST) three times, the mixture of the secondary antibodies was applied to the sections. The sections were placed at room temperature for 2 h, followed by washing three additional times with PBST. Fluorescence signals were detected with a Leica DM 2500 microscope.
Pavlovian Fear Conditioning
Quantitation
Male C57BL/6J mice at 2 months of age were used in this study. The procedure of fear conditioning was described previously (Hsiao et al., 2011). After a 120 s acclimation period, mice were presented with a tone (20 s, 80 dB, 5 kHz) that co-terminated with a foot shock (1 s, 0.7 mA). This tone–foot shock pairing was repeated 15 times with an inter-trial interval of 2 min. Between training sessions, 75% ethanol was used for cleaning. Cuedependent fear memory was assessed at 1, 14, and 42 days after training. Between each test session, 1% acetic acid was used for cleaning. The freezing behavior was defined as the absence of
The total numbers of BrdU-positive cells per section were counted in the amygdala, central amygdala nuclei, and medial amygdala nuclei. BrdU labeling was performed on every second section of 20 μm (microtome setting) in thickness, at least 560 µμm apart, from each mouse. Cells numbers were done by a MetaMorph image analysis system. Quantitative analysis of BrdU-immune fluoresccent-stained sections was done on a Leica DM 2500 microscope equipped with a video camera. One-way analysis of variance (ANOVA) and Newman–Keuls post hoc tests were performed to analyze the multiple comparisons. For double
Materials and Methods Animal Care
Hung et al. | 3
labeling, the colocalization of both BrdU and the markers NeuN, doublecortin (DCX), or Glial fibrillary acidic protein (GFAP) were confirmed on the slices using confocal laser scanning microscopy (Olmypus IX81, FV10-ASW). Triple labeling was performed for further verification of the BrdU positive cell phenotype. 4ʹ,6-diamidino-2-phenylindole (DAPI) nuclear labeling was performed on every second section, and the total number of Shhpositive cells was counted in the amygdala using a Leica DM 2500 microscope. To determine whether fear memory was specific to pairing groups, coronal sections of the amygdala from naive and unpaired groups were processed for Shh with DAPI labeling.
Antibodies for Staining The following primary antibodies were used: mouse monoclonal antibodies for BrdU (1:200, Millipore), NeuN (1:1 000, Millipore), and GFAP (1:1 000, eBioscience); rat monoclonal antibody for BrdU (1:100, Abcam) and Shh (1:100, R&D systems); rabbit polyclonal antibody for Shh (1:500, Millipore) and enhanced GFP (1:1000, Thermo Fisher Scientific); and rabbit monoclonal antibody for doublecortin (1:200, Cell Signaling Technology). The following secondary antibodies were used: Alexa Fluor®594-conjugated Goat-anti-rabbit IgG, Alexa Fluor®594-conjugated Donkey-anti-rat IgG, Alexa Fluor®488conjugated Sheep-anti-mouse IgG, Alexa Fluor®488-conjugated Goat-anti-rabbit IgG, Alexa Fluor®488-conjugated Donkey-antirat IgG, DyLightTM 405-conjugated Sheep-anti-mouse IgG, and DyLightTM 405-conjugated Donkey-anti-rat IgG (all 1:200, Jackson ImmunoResearch). DAPI (4’,6-diamidino-2-phenylindole; 1:1 000, Sigma-Aldrich) dye was used for staining of nuclei.
Western Blot Analysis Basolateral amygdala tissues were lysed by homogenization buffer (50 mM Tris-HCl, pH 7.5, 0.3 M sucrose, 5 mM EDTA), with protease/phosphatase inhibitor cocktail (Roche). Lysates were centrifuged at 12 000 rpm for 30 min. Supernatants were collected, and then protein concentration was measured by a Bradford assay. Proteins (150 μg) were taken and denatured at 95°C for 5 minutes. After electrophoresis via SDSpolyacrylamide gels (6.5–12.5%), proteins were transferred to nitrocellulose membranes. The membranes were blocked with 5% nonfat dry milk for 1 h at room temperature. Western blotting analysis used rabbit anti-Shh (1:3 000, Millipore), rabbit antiPtc1 (1:500, Proteintech Group), and rat anti-Gli1 (1:15 000, R&D systems) antibodies overnight at 4℃, and then incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. Signals were detected using Hyperfilm-ECL (Amersham Biosciences). The intensity was measured using Image-J.
Shh-shRNA Retrovirus Production To specifically knock down Shh in newborn cells, Shh-shRNA or scrambled shRNA was inserted into a pSIREN-RetroQ-ZsGreen1 retroviral shRNA expression vector (Clontech Laboratories, Inc.). The fluorescent ZsGreen1 protein was used as a tag to indicate transfection efficiency. Retroviruses were produced by the GP2-293 packaging cell line that cotransfected with the pSIREN-RetroQZsGreen1 retroviral shRNA expression vector and pVSV-G using Lipofectamine® LTX Reagent (Life Technologies). Retroviruses were harvested at 48 h after transfection, concentrated by Retro-X™ Concentrator (Clontech Laboratories), and purified to yield 1 × 106 transducing units/ml, which were stored at -80 ℃ until use. Viruses were stereotaxically injected into the amygdalas of the mice at the age of 2 months (anteroposterior, -1.6 mm; mediolateral, ±3.5 mm;
dorsoventral, -4.7 mm). The target sequence of Shh-shRNA and scrambled shRNA were described as follows: Shh-shRNA, 5×-CCCGAC ATCA TATTTAAGGAT-3×; and scrambled shRNA, 5×-GCTGTTGGACAG CGAGACCAT-3×.
Statistical Analysis All values in the text were mean ± standard error of the mean. Two-way ANOVA was used to analyze the differences in freezing responses tested at 1, 14, and 42 days after post-training between MAM- and Vehicle-, Ara-C- and Vehicle-, and retroviral vector (Retro-Shh-shRNA)- and Retro-scramble-shRNA-treated mice. Oneway ANOVA and Newman–Keuls post hoc comparisons were used to analyze the differences in BrdU+/NeuN+ cells and protein levels among naïve, unpaired, and paired mice. Unpaired t-tests were used to analyze differences of BrdU+/NeuN+ cells between Shh-shRNAtreated and scrambled control mice, and between MAM-treated and vehicle control mice. The level of significance was p
